Regulated nuclear transport is critical for the maintenance of the levels and activities of transcription factors, nuclear kinases, and replication factors. We identified Ca+2/calmodulin as an activator of nuclear import and suggested a role for Ca+2 in the regulation of nuclear import during cell activation. This evolutionarily ancient, calcium-dependent import pathway was proposed to facilitate uptake of a distinct subset of nuclear proteins during cell activation. The physiological significance of the Ca+2-calmodulin-regulated transport pathway became clear with the finding that defects in calmodulin-dependent nuclear import underlie certain forms of human sex reversal. The HMG-box architectural transcription factors SRY and SOX9 must enter the nucleus of Sertoli cells and bind tightly to target DNA for proper male gonad development. Thus, SRY acts as the primary trigger for maleness; nuclear transport of SRY is required to release this trigger. In a subset of human patients with autosomal sex reversal (Swyer?s syndrome, Campomelic dysplasia) nuclear transport of SRY and SOX9 is blocked. The mutations associated with these defects reside in a conserved calmodulin-binding motif near the amino terminus of SRY and SOX9. Thus, the SOX family of transcription factors appears to use Ca+2/calmodulin both as import receptor and molecular switch allowing for nuclear import and DNA binding. A similar autosomal sex reversal phenotype occurs when three insulin-related receptors are ablated in mice suggesting that intracellular signaling cascades may impact the normal functions of SRY and SOX9. More recently, the gene encoding TCF7L2 was shown to be a diabetes susceptibility locus in the Icelandic population. This transcription factor is a member of the HMG-box family containing a calmodulin-binding motif. The import of nuclear proteins by calmodulin, while functionally redundant with the canonic Ran-dependent pathway, is subject to independent regulation by intracellular Ca+2 mobilization. Abnormalities in calmodulin-dependent import emerge as the primary defect in patients with autosomal sex-reversal. These sex-reversal syndromes represent the first direct examples of a defect in a nuclear import pathway leading to human disease. In addition to analysis of the import of the HMG-box transcription factors in mammals, we have recently shown that the calmodulin-dependent nuclear import pathway functions in yeast. We are currently taking genetic approaches to define additional components of this novel import pathway and seek to identify small molecule inhibitors of the process.[unreadable] The nuclear pore is the conduit through which macromolecules move into and out of the nucleus. Nuclear pores are heavily modified by a unique posttranslational modification, O-GlcNAc. Addition and removal of O-GlcNAc are catalyzed by O-linked GlcNAc transferase (OGT) and O-GlcNAcase, respectively. Like nuclear pores, transcription complexes are also key targets for O-GlcNAc modification. Importantly, both histone deacetylases (HDAC) and OGT are recruited to Sin3a transcription-repression complexes. The C-terminus of the O-GlcNAcase has been shown to be a histone acetyltransferase (HAT). Therefore, O-GlcNAc appears to be a dynamic participant in histone remodeling complexes. Based on the targets modified by O-GlcNAc, we proposed that the enzymes of O-GlcNAc metabolism modulate nuclear transport, transcription, cell growth, and apoptosis in response to nutrient availability. O-GlcNAc is transferred to proteins from UDP-GlcNAc, a sugar nucleotide whose levels are regulated by the hexosamine biosynthetic pathway (HBP) acting as a cellular sensor of nutrient availability. By integrating these signals, the HBP regulates expression of a number of gene products that include leptin. In skeletal muscle, flux through the HBP correlates with the degree of insulin resistance. The HBP is also linked to pathways regulating cell proliferation and apoptosis; fibroblasts that cannot acetylate UDP-GlcNAc exhibit defects in proliferation, adhesiveness and resistance to apoptotic stimuli. Thus, by generating UDP-GlcNAc, the HBP may be viewed as a nutrient-sensing signaling pathway. We seek to determine how O-GlcNAc participates in this signaling cascade. [unreadable] We are testing the hypothesis that differentially targeted isoforms of the enzymes of O-GlcNAc metabolism mediate this glycan-dependent signaling pathway. By responding to nutrient levels, this pathway modulates gene expression, cell growth and programmed cell death. Examining the structure, targeting, and regulation of the enzymes of O-GlcNAc metabolism is our principal focus. We expressed fully functional OGT and O-GlcNAcase isoforms in E. coli. We recently solved the structure of the superhelical TPR (tetratricopeptide repeat) domain of OGT that mediates the recognition of target proteins and showed that exhibits structural similarities to importin alpha. Consistent with a role as a signaling molecule, we showed that OGT modifies glycogen synthase kinase-3 and casein kinase, two enzymes regulating glycogen synthesis. We also demonstrated that both isoforms of O-GlcNAcase are active enzymes modulating cellular O-GlcNAc levels. Mutational analysis of OGT and O-GlcNAcase allowed us to define catalytic domains. We showed that OGT isoforms are targeted to both nucleus and mitochondria. The differential localization of mitochondrial and nuclear isoforms of OGT argues that they perform unique intracellular functions in apoptosis and transcriptional repression respectively. O-GlcNAcase isoforms are also differentially targeted in cells; one isoform is nuclear while another accumulates at cellular sites of lipid storage. Small molecule inhibitors and substrates for the enzymes of O-GlcNAc metabolism are under development using both synthetic and natural product approaches.[unreadable] We have proposed that this intracellular glycan modification of Ser/Thr participates in diverse signaling pathways in a manner analogous to protein phosphorylation. Recently, the human O-GlcNAcase gene was identified as a non-insulin dependent diabetes mellitus (NIDDM) susceptibility locus in Mexican Americans. We seek to understand the biological functions of O-GlcNAc-dependent signaling and to determine whether altered O-GlcNAc metabolism contributes to human diseases such as diabetes mellitus and neurodegeneration. Transgenic overexpression of an isoform of OGT in muscle and fat induced Insulin resistance and Hyperleptinemia in mice. These data demonstrate a central role for OGT in the insulin and leptin-signaling cascades. The findings suggest a more general role for glycan-dependent signaling in nutrient sensing and the pathogenesis of NIDDM. Using reverse genetics, knockout, and other mouse transgenic models we are currently exploring the role of the enzymes of O-GlcNAc metabolism in signal transduction and the pathogenesis of diabetes mellitus. Caenorhabditis elegans is also an excellent model system in which to examine insulin signaling and neurodegeneration. We have identified viable Caenorhabditis elegans strains lacking OGT activity and O-GlcNAcase activity and have demonstrated that these knockout strains exhibit altered macronutrient storage and altered insulin-dependent dauer formation. This model should allow a genetic dissection of the role of hexosamine signaling in insulin resistance and provide clues to how hexosamine signaling may contribute to human maladies.